The E2 protein regulates transcription and replication of the papillomavirus genome as a stable episome. Its N-terminal domain binds cellular factors and complexes necessary for these functions. These cellular proteins are targeted to the viral genome by the E2 C-terminal domain, which binds with high affinity to specific sequences in the viral DNA. The goal of this grant proposal is to identify and characterize the cellular proteins and pathways through which E2 actuates its diverse functions. The first Aim investigates the activities of E2 during cell cycle, when E2 localizes the viral E1 DNA helicase to the origin in S phase, and in mitosis when E2 tethers the viral genome to host chromosomes, mediates its partitioning, and as we propose here, abrogates mitotic checkpoints that might otherwise restrict host cell viability. We demonstrate E2 interactions with several mitotic proteins that will be further characterized in this Aim. These protein- protein interactions reveal novel mechanisms for viral genome attachment to chromosomes during mitosis. Other interactions suggest a mechanism to transport the viral genome on the mitotic spindle. Another goal of this aim is to investigate the ability of E2 to inhibit mitotic checkpoints. In one example, we test the hypothesis that E2 inhibits cytokinesis, a function that is probably necessary for viral genome amplification in differentiated epithelial cells. Loss of these E2 functions following viral genome integration and disruption of E2 may represent an important step in neoplastic progression by oncogenic human papillomaviruses. Aim 2 continues our investigations of the mechanism of transcriptional regulation, with particular emphasis on how E2 overcomes chromatin repression in vivo. We test the hypothesis that E2 interactions with components of the Swi/Snf chromatin-remodeling complex are necessary for transcriptional activation and repression. These studies will broaden our understanding of the molecular functions of E2 in the viral replicative program. Lay Statement: Papillomaviruses cause benign infections that can become malignant, and are the etiologic agent of cervical cancer. The E2 protein controls essential functions of the virus. We propose to identify the proteins in the cell that interact with E2 and their functions. These are involved in fundamental process including gene expression and DNA replication. Understanding how E2 operates will provide insights into the biology of these pathways and infer how aberrations may occur that lead to the development of cancer.